Composition comprising ketone body and nicotinamide adenine dinucleotide modulator and methyl donor

ABSTRACT

The present invention provides a composition comprising a mixture of an exogenous ketone body, an exogenous NAD modulator and a methyl donor. Typically, exogenous NAD modulator is an exogenous nicotinamide adenine dinucleotide (NAD) precursor. The present invention also provides a method of using such a composition for treating various clinical conditions, including metabolic disorders and neurocognitive impairments. The compositions of the invention can also be used to improve human performance in various competitive or environmental conditions.

FIELD OF THE INVENTION

The present invention relates to a composition comprising a mixture ofan exogenous ketone body (“EK”), an exogenous nicotinamide adeninedinucleotide (“NAD”) modulator and a methyl donor. Typically, exogenousNAD modulator is an exogenous nicotinamide adenine dinucleotide (NAD)precursor. The present invention also relates to a method of using sucha composition for treating various clinical conditions, includingmetabolic disorders and neurocognitive impairments. The presentinvention further relates to a method of improving human performance invarious competitive or environmental conditions.

BACKGROUND OF THE INVENTION

Ketosis is a metabolic state in which some of the body's energy supplycomes from ketone bodies (e.g., acetone, acetoacetate andβ-hydroxybutyrate) in the blood. It is characterized by raised level ofketone bodies. Typically, serum concentration of ketone bodies is over0.5 mM in ketosis. Ketosis is pathological in certain conditions, suchas diabetes. However, ketosis can also be achieved using a diet that isvery low in carbohydrates, through prolonged fasting, or in intermittentfasting.

A number of clinical conditions can benefit from dietary ketosis, suchas epilepsy and other neurological conditions. There is also a growingbody of evidence that athletic performance can benefit from ketosisinduced by diet. This state of dietary ketosis is attainable, but theketogenic diets needed to achieve and/or to maintain ketosis are verydifficult to sustain. Thus, a source of safe and biologically activeexogenous ketone bodies may provide a solution for those seeking toachieve the metabolic state of ketosis, but who cannot or will notfollow the necessary restrictive diet to achieve ketosis.

A small number of ketone bodies have been developed for oral delivery,such as the salts of acetoacetate and β-hydroxybutyrate. However, theseketone bodies have largely suffered from poor oral tolerance and sideeffects. Some are unacceptably high in sodium, which significantlyoffsets any beneficial effects.

Ketosis is facilitated by nicotinamide adenine dinucleotide (“NAD”)modulator, which facilitates the conversion of other ketone bodies(e.g., BHB) to acetoacetate, which is one of the principal stepsrequired for conversion of the ketone bodies to a usable fuel(ultimately as acetyl-CoA). Regarding NAD upregulation, variants of theB vitamin analogue, nicotinamide riboside (“NR”), have been explored fortheir effects on NAD. NR raises blood and tissue NAD levels far abovethat of other B vitamin analogues (niacin, nicotinamide, nicotinic acid,nicotinamide mononucleotide).

Upregulation of NAD utilizes methyl donor. Therefore, the amount ofavailable methyl donor is expected to be reduced significantly due toincrease in NAD production resulting from administration of exogenousNAD modulator. This depletion in methyl donor can result in variousundesired side-effects.

To date, efforts to provide supplementary ketone bodies resulted in poortolerability of the exogenous ketone body (EK) supplement. Furthermore,all current EK supplements lack NAD modulator, which facilitates theconversion of other ketone bodies to acetoacetate. In addition,conventional supplements directed to upregulating NAD lacks methyl donorto offset the effects of methyl donor depletion.

Therefore, there is a need for a composition that can allow one toachieve the metabolic state of ketosis without the need for a strictdiet regime. Furthermore, there is a need for a composition thatincludes an EK supplement, an NAD modulator and a methyl donor.

SUMMARY OF THE INVENTION

The present invention provides a composition that includes an exogenousketone body (“EK”), an exogenous NAD modulator and a methyl donor. Insome embodiments, the exogenous ketone body comprises an ester of(R)-3-hydroxybutyrate, a derivative of an R-butyrate, a conjugate ofbutyrate, or a derivative thereof or a combination thereof. Exemplaryderivatives of beta-hydroxybutyrate (“BHB”) include esters of BHB, andcompounds where the hydroxyl group of BHB is bonded to a group that maybe cleaved in vivo to regenerate the free hydroxyl group, such asacetate, formate, benzoates and carbamates. Yet in other embodiments,the exogenous NAD modulator is an exogenous NAD precursor, which isconverted in vivo to an NAD modulator. Exemplary NAD modulators include,but are not limited to, nicotinoyl riboside, nicotinamide riboside,nicotinic acid mononucleotide, nicotinamide mononucleotide or aderivative thereof (such as β-nicotinamide ribose monophosphate), or acombination thereof.

The composition of the invention also includes a methyl donor. In oneparticular embodiment, the methyl donor comprises vitamin B₁₂, folate,S-adenosylmethionine, betaine, choline, or a combination thereof.

Another aspect of the invention provides a method for treating a subjectsuffering from a clinical condition associated with an elevated plasmalevel of free fatty acids. The method comprises administering to thesubject a therapeutically effective amount of a composition of theinvention.

Yet another aspect of the invention provides a method for suppressingappetite, treating obesity, promoting weight loss, maintaining a healthyweight or decreasing the ratio of fat to lean muscle in a subject. Themethod comprises administering to the subject in need thereof atherapeutically effective amount of a composition of the invention.

Still another aspects of the invention provide a method for treating acondition selected from muscle impairment and muscle fatigue. The methodcomprises administering to a subject in need thereof a therapeuticallyeffective amount of a composition of the invention.

Further aspect of the invention provides a method for treating traumaticinjury to the brain, including but not limited to traumatic brain injurydue to blast. The method comprises administering to a subject in needthereof a therapeutically effective amount of a composition of theinvention. In some embodiments, the traumatic brain injury comprises avascular injury to the brain. Exemplary vascular injuries to the braininclude stroke and ischemia.

Another aspect of the invention provides a method for improvingneurocognitive function in a subject. The method comprises administeringto a subject in need of such a treatment a therapeutically effectiveamount of a composition of the invention. In one particular embodiment,the method is used to treat a subject suffering from a mild cognitiveimpairment or Alzheimer's disease.

DETAILED DESCRIPTION OF THE INVENTION

Ketone bodies are chemical compounds that are produced by the liver fromfatty acids released from adipose tissue. Ketone bodies themselves canbe used as a source of energy in most tissues of the body. The intake ofcompounds that boost the levels of ketone bodies in the blood can leadto various clinical benefits, including an enhancement of physical andcognitive performance, and in the treatment of cardiovascularconditions, diabetes, neurodegenerative diseases and epilepsy.

Organs like the brain are fully capable of converting ketone bodies toATP. For example, for β-hydroxybutyrate (“BHB”), conversion to usefulenergy includes the steps of converting BHB to acetoacetate,acetoacetate to acetoacetyl CoA, and acetoacetyl CoA to acetyl CoA.Indeed, the amounts and activities of ketone body-metabolizing enzymesin organs such as the brain are not changed by glucose status and alwaysexceed the amount necessary to supply the brain's total energy needs.

Ketone bodies include (R)-3-hydroxybutyrate and acetoacetate. Thesecompounds could in theory be administered directly to achieve elevatedlevels of ketone bodies in a subject. However, direct administration ofthe compounds in high doses is unpractical and potentially dangerous.For example, direct administration of either (R)-3-hydroxybutyrate oracetoacetate in high doses of its free acid form can result insignificant ketoacidosis following rapid absorption from thegastrointestinal tract. Administration of the sodium salt of thesecompounds in unregulated amounts is also unsuitable, due to apotentially dangerous sodium overload that could accompanyadministration of therapeutically relevant amounts of the compounds.

Nicotinamide adenine dinucleotide (NAD+) is a natural coenzyme thatfunctions as an intermediary in cellular oxidation and reductionreactions, as well as an ADP-ribosyltransferase substrate. Alteringintracellular NAD+ levels can improve the health of a cell, butintroduction of compounds that enter NAD+ metabolic pathways can alsoprove toxic to cells (e.g., benzamide riboside (BAR)).

In some embodiments, compositions of the invention are capable ofimproving the health of damaged or diseased cells, for example, byaltering intracellular NAD+, NADH, NADP+ or NADPH levels. Moreover,compositions of the invention do not have any significant adverseeffects when administered therapeutically, i.e., side-effects areobserved in less than 10%, typically less than 5% and often less than 1%of the subject or patient.

In one particular embodiment, the composition of the invention includesan exogenous ketone body (“EK”), an exogenous NAD modulator and a methyldonor. In one embodiment, the compositions of the invention areformulated as a single unit. Thus, the exogenous ketone body, theexogenous NAD modulator and the methyl donor are intimately mixed in onesingle solid or liquid solution. Suitable exogenous ketone bodiesinclude an ester of β-hydroxybutyric acid, in particular an ester of(R)-3-hydroxybutyrate of the formula:

where R¹ is alkyl, hydroxyl alkyl, or a linker attached to aphospholipid, and R² is hydrogen, carboxylate (e.g., acetate, formate,benzoates and carbamates) or alkyl. Alkyl refers to a saturated linearmonovalent hydrocarbon moiety of one to twelve, typically one to eight,and often two to six, carbon atoms or a saturated branched monovalenthydrocarbon moiety of three to twelve, typically three to six, carbonatoms. Exemplary alkyl groups include, but are not limited to, methyl,ethyl, n-propyl, 2-propyl, tent-butyl, pentyl, and the like. The term“hydroxyl alkyl” refers to alkyl group having one or more, typicallyone, two or three hydroxyl (—OH) functional groups. Exemplary hydroxylalkyl groups include 3-hydroxylbutyl, 2-hydroxylbutyl,1-methyl-2-hydroxylpropyl, 1,3-butanediol, glycerol, glycol, etc. In oneparticular embodiment, R¹ is (R)-3-hydroxylbutyl. Still in anotherembodiment, R² is hydrogen. Yet in another embodiment, EK is glycerolesters of hydroxybutyric acid, such as those disclosed in U.S. Pat. No.5,693,850, issued to Birkhahn et al., which is incorporated herein byreference in its entirety. In particular, the glycerol can include one,two or three BHB's.

In one particular embodiment, EK is glycerol ester of BHB of thefollowing formula:

where each of R⁶, R⁷ and R⁸ is independently selected from the groupconsisting of hydrogen or BHB moiety of the formula —C(═O)CH₂CH(OR²)CH₃(where R² is H or a hydroxyl protecting group) or a hydroxyl protectinggroup; provided at least one of R⁶, R⁷ and R⁸ is BHB moiety. Suitablehydroxyl protecting groups includes hydroxyl protecting group asdiscussed herein for R², as well as those known to one skilled in theart of organic chemistry. See, for example, T. W. Greene and P. G. M.Wuts, Protective Groups in Organic Synthesis, 3^(rd) edition, John Wiley& Sons, New York, 1999; Harrison and Harrison et al., Compendium ofSynthetic Organic Methods, Vols. 1-8 (John Wiley and Sons, 1971-1996;chem.iitb.ac.in/˜kpk/pg.pdf; andfaculty.chemistry.harvard.edu/files/myers/files/7-protective_groups_.pdf,all of which are incorporated herein by reference in their entirety. Insome embodiments, hydroxyl protecting group is an acyl group, inparticular and acetyl group.

The amount of exogenous ketone body present in the composition of theinvention can range from about 1 mg to about 50,000 mg, typically fromabout 10,000 mg to about 25,000 mg, and often from about 15,000 mg toabout 20,000 mg. The term “about” when referring to a numeric valuemeans±20%, typically±10% and often±5% of the numeric value.

One particular aspect of the invention provides a composition where theEK (e.g., BHB or a derivative thereof or a combination thereof) is boundto a phospholipid, and methods for using and producing the same. In oneparticular embodiment, the EK is bound to a phospholipid that comprisesa fatty acid ester moiety that is derived from a substantiallynon-immunogenic C₄-C₂₂ fatty acid. In some embodiments, the C₄-C₂₂ fattyacid is an omega-3 fatty acid, an omega-6 fatty acid, an omega-9 fattyacid or a polyunsaturated fatty acid (“PUFA”). Exemplary fatty acidsthat are useful in the invention include, but are not limited to,docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), stearidonicacid (SDA), α-linolenic acid (ALA), eicosatetraenoic acid (ETA),γ-linolenic acid (GLA), dihomo-γ-linolenic acid (DGLA), calendic acid(CLA), and docosapentaenoic acid (DPA).

Still in other embodiments, the phospholipid further comprises choline,serine, inositol, ethanolamine, or other polar group. The phospholipidscan be derived from yeast, marine animal (such as krill), plant (such assunflower seed), and/or marine plant (such as microalgae). Thephospholipids can also be synthetically modified (e.g., catalyticallyre-randomized), e.g., via esterfication/transesterfication/acylation.Such synthetic modifications (e.g., re-randomization) can be used toprepare phospholipids comprising a wide variety of different fatty acidesters and/or a wide variety of combination of fatty acid esters.Furthermore, such synthetic modification also provides a wide variety ofpolar groups to be attached to the phosphate moiety of the phospholipid.Such synthetic modification can be achieved using an enzyme, a chemicalcatalyst, ultrasound, electromagnetic energy, or a combination thereofIn some embodiments, synthetic modification comprises reactingphospholipids with fatty acids, to modify (e.g., rearrange) one or moreof the terminal positions associated with the phospholipid, removing orhydrolyzing at least one fatty acid ester group to produce the hydroxylgroup, and covalently attaching (i.e., forming an ester group with) atleast one specific fatty acid of C₄ or higher, e.g. C₁₆, C₁₈, C₂₀, orC₂₂, to produce the fatty acid ester group.

Still other aspects of the invention provide liposome compositionscomprising a structured lipid. In some embodiments, the structured lipidcomprises a phospholipid including, but not limited to, compound offormula PL-1 discussed below. Still in other embodiments, the structuredlipid comprises a first fatty acid ester in which the first fatty acidis derived from a first lipid source, a second fatty acid ester in whichthe second fatty acid is derived from a second lipid source, and a thirdfatty acid ester in which the third fatty acid is derived from a thirdlipid source. In some particular embodiments, the second fatty acid isEPA and the third fatty acid is DHA. Still in other particularembodiments, the second fatty acid is ETA. Yet in other particularembodiments, the second fatty acid is ETA, EPA, or DHA.

In one particular embodiment of the invention. the phospholipid is ofthe formula:

where at least one of R³ and R⁴ is BHB or a derivative thereof (i.e.,CH₃—CH(OR²)—CH₂—C(═O)—, where R² is as defined herein), and the other isa C₄-C₂₂ fatty acid moiety (i.e., —C(═O)C₃-C₂₁ fatty acid); and R⁵together with the oxygen atom to which it is attached forms choline,serine, inositol, or ethanolamine moiety. In one particular embodiment,R³ is BHB or a derivative thereof and R⁴ is C₄-C₂₂ fatty acid. Yet inanother embodiment R³ is C₄-C₂₂ fatty acid and R⁴ is BHB or a derivativethereof. Still in another embodiment, both R³ and R⁴ are BHB or aderivative thereof, each of which is independent of the other (i.e., R²in R³ can be H and R² in R⁴ can be acetate, etc.). Suitably fatty acidsfor R³ or R⁴ include those described herein such as DHA, EPA, SDA, ALA,ETA, DPA, GLA, DGLA, and CLA.

In another embodiment, the C₄-C₂₂ fatty acid is an omega-3 fatty acid,an omega-6 fatty acid, or an omega-9 fatty acid.

Still in another embodiment, the C₄-C₂₂ fatty acid comprises α-linolenicacid (ALA), docosahexaenoic acid (DHA), eicosatetraenoic acid (ETA),eicosapentaenoic acid (EPA), stearidonic acid (SDA), γ-linolenic acid(GLA), calendic acid (CLA), docosapentaenoic acid (DPA), or acombination thereof.

Yet in other embodiments, the fatty acid is a C₁₆-C₂₂ fatty acid such asDHA, EPA, ETA, or a combination thereof

Exemplary exogenous NAD modulators that are useful in compositions ofthe invention include, but are not limited to, nicotinamide ribosidehalide or derivatives (collectively or individually sometimes referredto as “NR”), where halide can be iodide, bromide or chloride. Typically,halide is chloride. Nicotinamide riboside chloride has been shown inanimals and in humans to raise blood and tissue levels of NAD. Thus, NRrepresents an exogenous precursor to endogenous NAD formation. It isknown that the conversion of β-hydroxybutyrate to acetoacetate isNAD-dependent. Compositions of the invention combine exogenous ketonebody (such as BHB) and NAD modulator into a single unit. In someembodiments, NAD modulator is NR. Particularly useful NAD modulatorsinclude nicotinamide riboside, nicotinamide riboside chloride,nicotinamide mononucleotide or a combination thereof.

The term “derivatives” when referring to nicotinamide riboside refers tocompounds in which one or more of the hydroxyl groups are protected witha protecting group and can be cleaved in vivo to produce free hydroxylgroup. Exemplary protecting groups include acetyl group (CH₃C(═O)—),acetoacetyl group, formate, benzoates, carbamates, etc.

The amount of exogenous NAD modulator present in the composition of theinvention can range from about 0.001 mg to about 5,000 mg, typicallyfrom about 250 mg to about 1000 mg, and often from about 125 mg to about2000 mg.

Generally, the ratio between the exogenous ketone body and the exogenousNAD modulator in the composition of the invention ranges from about50,000,000:1 to about 10:1, typically from about 100:1 to about 25:1 andoften from about 160:1 to about 10:1.

Compositions of the invention not only raise blood ketone bodies byproviding an exogenous ketone body, but also include exogenous NADmodulator that facilitates or aid in conversion of ketone bodies toacetoacetate. When administered, the exogenous ketone body BHB isconverted to acetoacetate in vivo via the enzyme BHB dehydrogenase,which is modulated by the exogenous NAD modulator. BHB dehydrogenase isNAD-dependent. Thus, the exogenous NAD modulator upregulates endogenousNAD synthesis, which accelerates the conversion of BHB to acetoacetate.In this manner, more acetoacetate is readily available to be convertedto acetyl-Co-A for direct use as an energy substrate.

This combination of active agents (EK and NAD modulator) is applicableacross a range of work and performance states where greater energy froma non-lipid and non-carbohydrate substrate is desired. This combinationof active agents is also applicable across a range of clinicalconditions where greater energy from a non-lipid and non-carbohydratesubstrate is desired, where NAD production is low, where greater NADdemands exist, or where NAD is being consumed by other physiologicprocesses (such as PARP-1 activation).

Compositions of the invention are useful in elevating bloodconcentrations of ketone bodies when administered to a subject, as wellas increasing blood and tissue levels of NAD. By using exogenous NADmodulator (e.g., NR) to raise NAD to facilitate conversion of exogenousketone bodies to acetoacetate, it is believed that exogenous NADmodulator use is accompanied by elevated plasma methylnicotinamide(methylated nicotinamide), increased methylnicotinamide excretion inurine, elevated nuclear uracil, and elevated serum, plasma, or wholeblood homocysteine. Typically, the methyl groups needed to formmethylnicotinamide are derived from a small pool of endogenous methyldonors. The amount of available methyl donor is expected to be reducedsignificantly due to increase in NAD production resulting fromadministration of exogenous NAD modulator and the resultant methylationof nicotinamide. Thus, compositions of the invention also include anexogenous methyl donor. Exemplary exogenous methyl donor that can beused in compositions of the invention include, but are not limited to,vitamin B₁₂, folate, betaine, choline, or a derivative thereof or acombination thereof. Without being bound by any theory, it is believedthat inclusion of exogenous methyl donor in compositions of theinvention allows maintaining of the endogenous methyl donor pool and/orprevention of methyl pool depletion.

Compositions of the invention are useful in elevating bloodconcentrations of ketone bodies when administered to a subject, as wellas increasing blood and tissue levels of NAD. By using exogenous NADmodulator to raise NAD to facilitate conversion of exogenous ketonebodies to acetoacetate, it is believed that exogenous NAD modulator useis accompanied by inhibition of histone deacetylases (HDAC).

Normally, the tails of histone proteins are positively charged, due totheir lysine and arginine residues. These positive charges allow them tointeract with the negative charges on the deoxyribonucleic acid (DNA)backbone. When DNA and histones are bound together, the DNA-dependentRNA polymerase (DdRP) cannot make contact with the DNA. This contactinhibition reduces or prevents transcribing of the code inside the DNA,thereby reducing or preventing production of messenger RNA, andultimately results in reduction or prevention of protein translation.

Acetylation of histone (i.e., covalent attachment of acetyl groups tothe lysine and arginine residues) eliminates the positive charges onlysine and arginine and reduces histone-DNA binding. This acetylation ofhistone results in the DNA to unwind from the histone protein complexesand makes DNA available for transcription. This acetyl addition processis catalyzed by histone acetyltransferases (HATs). Reduced HAT activitycorrelates with lower global acetylation and transcriptional dysfunctionin multiple diseases. Histone deacetylases (HDACs) are a class ofprotein that catalyze the removal of the acetyl groups from inter aliahistone, allowing for DNA to once again interact with histone complexes,and prevent transcription. Thus, HDACs play an important role in theregulation of gene transcription. Histone deacetylate inhibitors (HDIs)have been widely used in psychiatric care and are potentially useful intreating a large number of other disease states, such asneurodegenerative diseases, diabetes, and cancer.

Aberrant hypermethylation is believed to be one of the major mechanismsin carcinogenesis and some critical growth regulatory genes have showncommonality in methylation across solid tumors. Compositions of theinvention are useful in epigenetic modification via NR reducing genepromoter methylation and the ketone component blocking histonedeacetylase (HDAC). Exogenous ketone bodies such as BHB are HDACinhibitors. Studies have shown nicotinamide adenine dinucleotide (“NAD”)regulates gene methylation. Therefore, compositions of the invention arealso useful in clinical conditions associated with disordered genemethylation and acetylation of histone.

Compositions of the invention can also be used to lower homocysteine,restoring S-adenosylmethionine, reducing nuclear uracil, and/orpreserving the methyl pool.

The exogenous ketone body as a source of acetyl Co-A can also be used topreserve NAD for use in PARP-directed DNA repair. The presence ofexogenous ketone body, such as BHB, results in consumption of fewer NAD+per acetyl-CoA produced. Metabolism of one molecule of glucose to twomolecules of acetyl-CoA involves conversion of four molecules of NAD+into NADH. Two of these are converted in the cytosol during glycolysis,and two in the mitochondrion by pyruvate decarboxylase. The cytosolicNADHs are shuttled into mitochondria, leading to depletion of thecytoplasmic NAD pool with high glucose utilization. By contrast,metabolism of one BHB molecule into the same two molecules of acetyl-CoAinvolves conversion of only two molecules of NAD+ into NADH, both in themitochondrion by BDH1 and thereby preserving the cytoplasmic NAD pool.The cytoplasmic and mitochondrial NAD pools are relatively distinct;therefore, the preservation of cytoplasmic NAD+ by BHB may haveimportant cellular effects. NAD+ is a cofactor for sirtuin deacylases(such as nuclear/cytoplasmic SIRT1) as well as poly-ADP-ribosepolymerase (PARP). Consumption of NAD+ by PARP or overproduction of NADHmay promote age-related diseases by inhibiting the activity of sirtuins.Conversely, repletion of NAD+ by exogenous feeding with nicotinamidemononucleotide has been shown to improve glucose tolerance in bothhigh-fat diet-fed and aged mice. The relative sparing of NAD+ byutilization of BHB vis a vis glucose may therefore have importantconsequences for metabolic diseases and diabetes.

It has been shown that in some instances hypoxia results in increasedPARP and DNA polymerase activity, for example, in cerebral corticalneuronal nuclei to repair the hypoxia-induced damage to genomic DNA. Inaddition, high-altitude illness is caused primarily by hypobaric hypoxia(HH), which is an acute physiological stressor that impacts the centralnervous system, resulting in several physiological adaptations. Ingeneral, the severity and duration of the symptoms vary, depending onthe altitude and rate of ascent, often persisting after returning tolower altitudes. The development of these symptoms has been linked tohypoxia-induced oxidative and nitrosative stress. HH promotes anincrease in NO production, which correlates with PARP activation.Specifically, PARP-1 detects DNA-strand breaks caused by genotoxicagents, such as peroxynitrite and reactive oxygen species (ROS)associated with hypoxia. Hypoxia-inducible factor(s) (HIF-1 and HIF-2)are transcription factors with a central role in the accommodation ofhypoxia. PARP-1 forms a physical complex with HIF-2, which promotes theexpression of HIF-2 mediated genes. Thus, PARP-1 activation facilitatesHIF-1 and HIF-2 activation, which subsequently contributes to thedownregulation of mitochondrial activity. The downregulation ofmitochondrial activity is concomitant with the depletion of NAD+, whichis triggered by PARP assembly.

Obstructive sleep apnea (OSA) is associated with inter alia intermittenthypoxia, PARP activation, lower intra-epidermal nerve fiber density(IENFD), peripheral neuropathy (DPN), and diabetic foot ulceration(DFU), which may occur in the presence of type 2 diabetes. Theintermittent hypoxia of OSA may also occur in obese or non-obese states.PARP activation is a potential mechanism linking OSA to DPN andendothelial dysfunction in those with obesity, metabolic syndrome, ortype 2 diabetes.

Compositions of the invention can also be used to reduce the adverseeffects of PARP activation due to hypoxia or intermittent hypoxia byproviding a non-glycogenic energy substrate, an NAD+ precursor (NR), andmethyl donors. Thus, compositions of the invention can be used toprevent the ATP depletion associated with PARP activation withoutadversely affecting methyl donor pool (such as S-adenosylmethionine,betaine, choline, B₁₂, folate). Exemplary clinical conditions that canbe treated by reducing/preventing ATP depletion associated with PARPactivation include altitude-related hypobaric hypoxia, obstructive sleepapnea, endothelial dysfunction, lower intra-epidermal nerve densityloss, diabetic peripheral neuropathy, and diabetic foot ulceration.

The amount of exogenous methyl donor, as vitamin B12 or folate, presentin the composition of the invention can range from about 0.0001 mg toabout 20 mg, typically from about 400 mcg to about 5 mg, and often fromabout 800 mcg to about 10 mg.

The amount of exogenous methyl donor, as betaine, present in thecomposition of the invention can range from about 0.001 mg to about5,000 mg, typically from about 150 mg about 500 mg, and often from about500 mg to about 1,500 mg.

The amount of exogenous methyl donor, as choline, present in thecomposition of the invention can range from about 0.001 mg to about2,000 mg, typically from about 100 mg to about 400 mg, and often fromabout 25 mg to about 500 mg.

The amount of exogenous methyl donor, as S-adenosylmethionine, presentin the composition of the invention can range from about 0.001 mg toabout 2,000 mg, typically from about 100 mg to about 500 mg, and oftenfrom about 25 mg to about 500 mg.

In some embodiments, the compositions of the invention also includecholine. Exemplary forms of choline that can be used in compositions ofthe invention include, but are not limited to, choline citrate(Citicholine), choline bitartrate, phosphatidylcholine, alpha-GPC(L-Alpha Glycerylphosphorylcholine), CDP choline (cytidine5′-diphosphocholine) and the like.

Exemplary betaine that can be used in composition of the inventionincludes, but is not limited to, betaine monohydrate, betaine anhydrous,trimethylglycine (TMG) and the like.

Exemplary forms of vitamin B₁₂ that can be used in compositions of theinvention include, but are not limited to, cyanocobalamin,methylcobalamin, hydroxocobalamin, adenosylcobalamin and the like.

Exemplary forms of folate that can be used in compositions of theinvention include, but are not limited to, folinic acid,5-methyltetrahydrofolate, L-methylfolate, L-methylfolate calcium and thelike.

Exemplary forms of S-adenosylmethionine that can be used in compositionsof the invention include, but are not limited to, S-adenosylmethionine.

In some embodiments, the ratio of the exogenous NAD modulator to theexogenous methyl donor in the composition of the invention ranges fromabout 5,000:1 to about 1:1, typically from about 500:1 to about 3:1 andoften from about 1000:1 to about 2:1. In other embodiments, the ratio ofthe exogenous NAD modulator to the exogenous methyl donor as vitamin B₁₂or folate ranges from about 6.25:1 to about 50,000,000:1, typically fromabout 25:1 to about 2500:1 and often from about 25:1 to about 1250:1.Still in other embodiments, the ratio of the exogenous NAD modulator tothe exogenous methyl donor as betaine ranges from about 1:1 to about5,000,000:1, typically from about 1.667:1 to about 6.667:1 and oftenfrom about 0.25: 1 to about 1.333:1. Yet in other embodiments, the ratioof the exogenous NAD modulator to the exogenous methyl donor as cholineranges from about 1:1 to about 5,000,000:1, typically from about 2.5:1to about 10:1 and often from about 5:1 to about 80:1. In otherembodiments, the ratio of the exogenous NAD modulator to the exogenousmethyl donor as S-adenosylmethionine ranges from about 1:1 to about5,000,000:1, typically from about 0.625:1 to about 10:1 and often fromabout 5: 1 to about 80:1.

Compositions of the invention can be used for other indications inaddition to those disclosed above. In one embodiment, compositions ofthe invention can be used to increase the lifespan of a cell or asubject. Compositions of the invention can also be used in treatingand/or preventing a wide variety of diseases and disorders including,for example, diseases or disorders related to aging or stress, diabetes(type I or type II), obesity, neurodegenerative diseases (such asAlzheimer's disease and neurocognitive impairment, etc.), cardiovasculardisease, muscular disorders, blood clotting disorders, inflammation,cancer, eye disorders, or to promote alertness or improve cognitivefunction, to treat or improve hypoxic conditions, or to improve athleticperformance, or to improve work capacity in an extreme environment orwork condition.

Compositions of the invention can also be used to treat or prevent oneor more of cataracts, retinopathy, retinitis pigmentosa, ocular neuritisor a vascular disease of the capillary beds of the eye.

In addition, compositions of the invention are useful in reducing theweight of a subject, preventing weight gain in a subject and/orincreasing the ratio of lean muscle/fat ratio in a subject.

Compositions of the invention are also useful in preventing a subjectfrom acquiring insulin insensitivity or treating or preventing insulinresistance disorders.

In some embodiments, compositions of the invention consist essentiallyof EK, NAD modulator, a methyl donor and optionally a pharmaceuticallyacceptable carrier or excipient.

As discussed above, compositions of the invention comprising EK, NADmodulator and a methyl donor can be used to reduce the adverse effectsof PARP activation, such as preventing ATP depletion associated withPARP activation. Compositions of the invention are particularly usefulin reducing the adverse effects of PARP activation due to the presenceof a methyl donor. By including a methyl donor, compositions of heinvention do not adversely affect in vivo (i.e., naturally occurring)methyl donor pool (such as S-adenosylmethionine, betaine, choline, B₁₂,folate), i.e., the natural amount of methyl donor is not depletedsignificantly (about 20% of less, typically about 10% of less, and oftenabout 5% or less).

Specific examples of clinical conditions that can be treated byreducing/prevention ATP depletion associated with PARP activation usingcompositions of the invention include, but are not limited to, aneurodegenerative disease, stroke, Alzheimer's disease, dementia,cognitive impairment, Huntington's disease, Parkinson's disease,multiple sclerosis, diseases of myelin, mitochondrial disorders, muscleweakness, fatigue, depression, anxiety, age-related hearing loss,hypoxia, hyperoxia, altitude-related hypobaric hypoxia, obstructivesleep apnea, diabetes, endothelial dysfunction, lower intra-epidermalnerve density loss, peripheral neuropathy, diabetic foot ulceration,non-alcoholic fatty liver disease, traumatic brain injury, and epilepsy.This includes the effects of radiation associated with occupationalexposure, military combat, terrorism, nuclear accidents, space flight,medical treatment, and others. Exemplary forms of radiation that triggerDNA damage and repair include, electromagnetic radiation, such asinfrared, ultraviolet, radio waves, visible light, x-rays, and gammaradiation (γ); particle radiation, such as alpha radiation (α), betaradiation (β), and neutron radiation (particles of non-zero restenergy); solar energetic particle radiation (energetic electrons,protons, alpha particles, and heavier particles); acoustic radiation,such as ultrasound, sound, blast waves, shock waves, and seismic waves(dependent on a physical transmission medium); gravitational radiation,radiation that takes the form of gravitational waves.

Compositions of the invention can also be used to prevent brainN-acetylaspartate (NAA) depletion associated with PARP activationwithout adversely affecting methyl donor pool (e.g.,S-adenosylmethionine, betaine, choline, B₁₂, folate). Exemplary clinicalconditions that can be treated by preventing NAA depletion associatedwith PARP activation include stroke, Alzheimer's disease, dementia,cognitive impairment, Huntington's disease, multiple sclerosis, diseasesof myelin, mitochondrial disorders, muscle weakness, fatigue,depression, anxiety, age-related hearing loss, and epilepsy. DiminishedNAA is considered a marker for neuronal injury, morbidity, or metabolicdysfunction. Traumatic brain injury (TBI) results in a reduction inbrain NAA levels. NAA depletion is also noted in brain injury due totherapeutic radiation of brain tumors.

It should be noted that when the methyl donor pool is preserved byincluding a methyl donor in compositions of the invention, accumulationof uracil (U) in the nucleus is prevented or is significantly reduced.For example, if NAD modulator is used without a methyl donor, it willresult in increased U concentration in the nucleus as reduction in themethyl donor prevents conversion of uracil (U) to thymine (T). Increasein uracil concentration in nucleus can lead to a wide variety ofundesired side-effects including, but not limited to, single or doublestrand breaks in DNA, and reduced ability of cells to repair DNA damageeven with PARP activation. By providing a methyl donor, use ofcompositions of the invention preserves thymine synthesis, DNAstability, and the further potentially adverse effect on needing DNArepair enzymes, such as uracil glycosylases.

Compositions of the invention comprising EK, NAD modulator and a methyldonor can also be used to reduce the adverse effects of PARP activation,such as dysregulated ATP metabolism, lactate metabolism, and aberrantcell growth without adversely affecting methyl donor pool (such asS-adenosylmethionine, betaine, choline, vitamin B₁₂, folate). Exemplaryclinical conditions that can be treated by reducing/prevention ATP andlactate dysregulation associated with PARP activation include cancer.Glucose is a substrate for cancer cell biomass via glycolysis andconversion to lactate. Tumor cells cannot metabolize ketone bodiesreadily. Therefore, by providing ketone bodies as a fuel for cancertherapy and restricting glucose consumption, compositions of theinvention can also be used to treat cancer.

In some embodiments, the presence of EK protects against ionizingradiation. Accordingly, compositions of the invention can also be usedin treating cancer, reducing the side-effects of chemotherapy and/orradiotherapy.

Without being bound by any theory, it is believed that compositions ofthe invention also increase the number of cellular mitochondria in asubject. This increase results in higher energy expenditure resulting inweight loss and/or decrease in the total amount of fat in a subject.Accordingly, in some embodiments, compositions of the invention areuseful in preservation, restoration, or resynthesis of glycogen. Inother embodiments, compositions of the invention are useful inincreasing performance and endurance such as in athletics and otherstressful situations such as in combats.

In general, the compositions of the invention are administered in atherapeutically effective amount by any of the accepted modes ofadministration for agents that serve similar utilities. Generally,compositions of the invention are administered as nutraceutical orpharmaceutical formulations including those suitable for oral (includingbuccal and sub-lingual), administration. Typical manner ofadministration is generally oral using a convenient daily dosageregimen.

Compositions of the invention, together with one or more conventionaladjuvants, carriers, or diluents, can be placed into unit dosages. Thecompositions and unit dosage forms can be comprised of conventionalingredients in conventional proportions, with or without additionalnutraceutical compounds or principles. The compositions of the inventioncan be employed as solids, such as tablets or filled capsules,semisolids, powders, sustained release formulations, or liquids such assolutions, suspensions, emulsions, elixirs, or filled capsules for oraluse.

The compositions of the invention can be formulated in a wide variety oforal administration dosage forms. The compositions and dosage forms cancomprise one or more nutraceutically acceptable carriers and can beeither solid or liquid. Solid form preparations include powders,tablets, pills, capsules, cachets, and dispersible granules. A solidcarrier can be one or more substances which can also act as diluents,flavoring agents, solubilizers, lubricants, suspending agents, binders,preservatives, tablet disintegrating agents, or an encapsulatingmaterial. In powders, the carrier generally is a finely divided solidmixture which includes a finely divided active component. In tablets,the compositions of the invention generally are mixed with the carrierhaving the necessary binding capacity in suitable proportions andcompacted in the shape and size desired.

It should be appreciated other nutrients such as vitamins, mineralincluding trace minerals can also be included in compositions of theinvention.

When formulated as a unit dosage form, compositions of the invention canalso include one or more suitable carriers. Suitable carrier include butare not limited to magnesium carbonate, magnesium stearate, talc, sugar,lactose, pectin, dextrin, starch, gelatine, tragacanth, methylcellulose,sodium carboxymethylcellulose, a low melting wax, cocoa butter, and thelike. The term “preparation” or “unit dosage” is intended to include theformulation of the compositions of the invention with encapsulatingmaterial as carrier, providing a capsule in which the compositions ofthe invention, with or without carriers, is surrounded by a carrier,which is in association with it. Similarly, cachets and lozenges areincluded. Tablets, powders, capsules, pills, cachets, and lozenges canbe as solid forms suitable for oral administration.

Other forms suitable for oral administration include liquid formpreparations including emulsions, syrups, aqueous solutions, aqueoussuspensions, or solid form preparations which are intended to beconverted shortly before use to liquid form preparations. Emulsions canbe prepared in solutions, for example, in aqueous propylene glycolsolutions or may contain emulsifying agents, for example, such aslecithin, sorbitan monooleate, or acacia. Aqueous solutions can beprepared by dissolving the compositions of the invention in water andadding suitable colorants, flavors, stabilizers, and thickening agents.Aqueous suspensions can be prepared by dispersing the finely dividedcompositions of the invention in water with viscous material, such asnatural or synthetic gums, resins, methylcellulose, sodiumcarboxymethylcellulose, and other well-known suspending agents. Solidform preparations include solutions, suspensions, and emulsions, and cancontain, in addition to the compositions of the invention, colorants,flavors, stabilizers, buffers, artificial and natural sweeteners,dispersants, thickeners, solubilizing agents, and the like.

The nutraceutical preparations are typically in unit dosage forms. Insuch form, the preparation is often subdivided into unit dosescontaining appropriate quantities of the active component (e.g.,exogenous ketone body, exogenous NAD modulator and methyl donor). Theunit dosage form can be a packaged preparation, the package containingdiscrete quantities of preparation, such as packeted tablets, capsules,and powders in vials or ampoules. Also, the unit dosage form can be acapsule, tablet, cachet, or lozenge itself, or it can be the appropriatenumber of any of these in packaged form.

When it is possible that, for use in therapy, therapeutically effectiveamounts of a composition of the invention, as well as pharmaceuticallyacceptable salts thereof, can be administered as the raw chemical, it ispossible to present the active ingredient (i.e., exogenous ketone body,exogenous NAD modulator and methyl donor) as a nutraceutical orpharmaceutical composition. Accordingly, the disclosure further providesnutraceutical and/or pharmaceutical compositions, which includetherapeutically effective mounts of active ingredients (i.e., exogenousketone body, exogenous NAD modulator and in some embodiments optionallypresent methyl donor), and one or more nutraceutically orpharmaceutically acceptable carriers, diluents, or excipients. Whenapplied to a combination, the term refers to combined amounts of theactive ingredients that result in the therapeutic effect, whetheradministered in combination, serially, or simultaneously. Thecarrier(s), diluent(s), or excipient(s) must be acceptable in the senseof being compatible with the other ingredients of the formulation andnot deleterious to the recipient thereof. In accordance with anotheraspect of the disclosure there is also provided a process for thepreparation of a nutraceutical or pharmaceutical formulation includingadmixing the active ingredients with one or more nutraceutically orpharmaceutically acceptable carriers, diluents, or excipients.

Additional objects, advantages, and novel features of this inventionwill become apparent to those skilled in the art upon examination of thefollowing examples thereof, which are not intended to be limiting. Inthe Examples, procedures that are constructively reduced to practice aredescribed in the present tense, and procedures that have been carriedout in the laboratory are set forth in the past tense.

EXAMPLES

The mixture of exogenous NAD modulator, exogenous ketone, and exogenousmethyl donor(s) are incorporated into an oral dosage formulationincluding, but not limited to, a stabilized gel, liquid formulation,and/or bulk powder; which may or may not be incorporated into a foodmatrix, with a unit dosage set within the range set forth herein. Theunit dosage form is incorporated with the appropriate excipients asneeded, which is dictated by the set formulation.

The foregoing discussion of the invention has been presented forpurposes of illustration and description. The foregoing is not intendedto limit the invention to the form or forms disclosed herein. Althoughthe description of the invention has included description of one or moreembodiments and certain variations and modifications, other variationsand modifications are within the scope of the invention, e.g., as may bewithin the skill and knowledge of those in the art, after understandingthe present disclosure. It is intended to obtain rights which includealternative embodiments to the extent permitted, including alternate,interchangeable and/or equivalent structures, functions, ranges or stepsto those claimed, whether or not such alternate, interchangeable and/orequivalent structures, functions, ranges or steps are disclosed herein,and without intending to publicly dedicate any patentable subjectmatter. All references cited herein are incorporated by reference intheir entirety.

1. A composition comprising: (i) a therapeutically effective amount ofan exogenous ketone body present in the composition in an amount from 1mg to 50,000 mg, wherein the exogenous ketone body comprises3-hydroxybutyrate (“BHB”) or an ester of BHB; (ii) an exogenousnicotinamide adenine dinucleotide (“NAD”) modulator; and (iii) a methyldonor; wherein the ratio (w/w) of the exogenous ketone body to theexogenous NAD modulator is from 160:1 to 10:1; and wherein thecomposition is in a single unit form.
 2. The composition of claim 1,wherein the methyl donor is selected from the group consisting ofcholine, betaine, folate, vitamin B12, or S-adenosylmethionine.
 3. Thecomposition of claim 2, wherein the methyl donor is betaine.
 4. Thecomposition of claim 3, wherein the betaine is present in thecomposition in an amount from 0.001 mg to 5,000 mg.
 5. The compositionof claim 4, wherein the betaine is present in the composition in anamount from 150 mg to 1500 mg.
 6. The composition of claim 3, whereinthe ratio (w/w) of the exogenous NAD modulator to betaine ranges from0.25:1 to 6.667:1.
 7. (canceled)
 8. The composition of claim 2, whereinthe exogenous ketone body comprises BHB.
 9. The composition of claim 2,wherein the exogenous ketone body comprises an ester of BHB.
 10. Thecomposition of claim 9, wherein the ester of BHB is of the formula:

wherein each of R⁶, R⁷, and R⁸ is independently selected from the groupconsisting of hydrogen, BHB moiety or an acyl group; provided at leastone of R⁶, R⁷, and R⁸ is BHB moiety of the formula —C(═O)CH₂CH(OR²)CH₃,wherein R² is H or a hydroxyl protecting group.
 11. The composition ofclaim 2, wherein the exogenous NAD modulator is nicotinoyl riboside,nicotinamide riboside, nicotinic acid mononucleotide, nicotinamidemononucleotide, β-nicotinamide ribose monophosphate, or a combinationthereof.
 12. The composition of claim 11, wherein the exogenous NADmodulator is nicotinamide riboside, and wherein the nicotinamideriboside comprises a protecting group comprising an acetyl group(CH3C(═O)—), an acetoacetyl group, formate, benzoate, or carbamate. 13.The composition of claim 2, wherein the exogenous NAD modulator ispresent in the composition in an amount from 0.001 mg to 5,000 mg. 14.The composition of claim 2, wherein the exogenous NAD modulator ispresent in the composition in an amount from 125 mg to 2000 mg.
 15. Thecomposition of claim 2, wherein the ratio (w/w) of exogenous NADmodulator to methyl donor is between 0.25:1 to 6.667:1.
 16. (canceled)17. The composition of claim 1, further comprising a carrier.
 18. Thecomposition of claim 17, wherein the carrier is a liquid.
 19. Thecomposition of claim 17, wherein the composition is an emulsion, syrup,aqueous solution, or aqueous suspension.
 20. The composition of claim17, wherein the carrier comprises one or more of propylene glycol,lecithin, sorbitan monooleate, acacia, thickening agents, natural orsynthetic gums, resins, methylcellulose, flavors, sweeteners, colorants,or sodium carboxymethylcellulose.
 21. The composition of claim 17,wherein the composition is a solid.
 22. The composition of claim 21,wherein the composition is formulated as a powder, tablet, pill,capsule, cachet, lozenge, or as dispersible granules.
 23. Thecomposition of claim 21, wherein the composition comprises one or moreof a diluent, flavoring agent, solubilizer, lubricant, suspending agent,binder, preservative, tablet disintegrating agent, or encapsulatingmaterial.
 24. The composition of claim 10, wherein: the exogenous NADmodulator is nicotinoyl riboside, nicotinamide riboside, nicotinic acidmononucleotide, nicotinamide mononucleotide, β-nicotinamide ribosemonophosphate, or a combination thereof, wherein the exogenous NADmodulator is present in the composition in an amount from 0.001 mg to5,000 mg; and wherein the methyl donor is betaine, wherein betaine ispresent in the composition in an amount from 0.001 mg to 5,000 mg. 25.The composition of claim 24, wherein betaine is present in thecomposition in an amount from 150 mg to 1500 mg.
 26. The composition ofclaim 25, wherein the ratio (w/w) of the exogenous NAD modulator tobetaine ranges from 0.25:1 to 6.667:1.
 27. (canceled)
 28. (canceled) 29.The composition of claim 1, wherein the exogenous ketone body is BHB,and the BHB is in its free acid form.